EP3141908B1 - Reactifs de diagnostic - Google Patents

Reactifs de diagnostic Download PDF

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Publication number
EP3141908B1
EP3141908B1 EP16188822.7A EP16188822A EP3141908B1 EP 3141908 B1 EP3141908 B1 EP 3141908B1 EP 16188822 A EP16188822 A EP 16188822A EP 3141908 B1 EP3141908 B1 EP 3141908B1
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animal
seq
tuberculosis
diagnostic reagent
amino acid
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EP3141908A1 (fr
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Gareth Jones
Hans Vordermeier
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UK Secretary of State for Environment Food and Rural Affairs
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UK Secretary of State for Environment Food and Rural Affairs
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/5695—Mycobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0006—Skin tests, e.g. intradermal testing, test strips, delayed hypersensitivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/715—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons
    • G01N2333/7156—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons for interferons [IFN]

Definitions

  • the present invention relates to reagents for use in the detection of tuberculosis infections, particularly tuberculosis in mammals such as human beings and cattle, more particularly infection by Mycobacteria such as M. tuberculosis and M. bovis.
  • the reagents are useful to differentiate between animals with a tuberculosis infection and those which have been vaccinated against infection, as a positive result is only obtained from infected animals (or animals exposed to an infectious agent).
  • M. tuberculosis and M. bovis are important pathogens of man and animals. M. tuberculosis is thought to infect up to a third of the world's human population, remaining undetected during a latent phase of infection and reactivating to cause 10 million cases of tuberculosis and other diseases per year, resulting in 2 million deaths ( Corbett et al. (2003) Arch. Intern. Med. vol. 163 pp 1009-1021 ). M. bovis, which has more than 99.9% sequence identity with M. tuberculosis, is the predominant causative agent of bovine tuberculosis (BTB) and also causes disease in human. Cases of bovine tuberculosis in cattle caused by M.
  • BTB bovine tuberculosis
  • tuberculosis have also been reported, particularly in developing countries with high incidence rates of human TB (see, for example, Berg et al.(2009) PLoS ONE vol. 4 e5068 ).
  • BTB represents a significant economic burden to the agricultural industries of various countries including the United Kingdom ( Krebs (1997) "Bovine Tuberculosis in Cattle & Badgers" HMSO, London, United Kingdom ).
  • the primary diagnostic test used in the control and surveillance of bovine TB is the tuberculin skin-test, a test that has remained in the forefront of TB diagnosis in both man and cattle for over 100 years.
  • the development of the test arose following the preparation of the first 'tuberculin' by Robert Koch in 1890. Whilst Koch's tuberculin failed to live up to its initial claims of having curative properties, its diagnostic potential was quickly realised.
  • the most common formats of the test used in cattle are the caudal fold test (CFT), the single intradermal cervical tuberculin test (SIT) and the single intradermal comparative cervical tuberculin test (SICCT) ( Monaghan et al. (1994) Vet. Microbiol. vol. 40 pp 111-24 ).
  • test formats use a purified protein derivative (PPD) tuberculin prepared from a culture of M. bovis (PPD-B) as the primary diagnostic antigen.
  • PPD purified protein derivative
  • the SICCT test includes the use of a M. avium derived PPD (PPD-A) to provide a measure of environmental sensitisation. It is the more specific of the two tests ( Plum (1931) Cornell Vet. vol. 21 pp 68-76 ; Stenius (1938) Veterinary Record vol. 50 pp 633-7 ) and is therefore the adopted test format in the UK.
  • IFN- ⁇ interferon gamma
  • mice with a disrupted IFN- ⁇ gene and humans with mutated IFN- ⁇ receptor are highly susceptible to mycobacterial infections.
  • specificity constraints are associated with the use of PPD in such assays. These arise due to the crude mixture of M. bovis proteins that PPD contains, many of which are cross-reactive with the BCG vaccine strain and environmental mycobacterial species such as M. avium and M. intracellulare.
  • the term "tuberculosis infection assay" used in the present specification may refer to any of these diagnostic tests referred to above.
  • Bovine TB is a significant and ongoing problem in the UK (http://www.defra.gov.uk/food-farm/animals/diseases/tb, accessed 14 th July 2011).
  • Cattle vaccination has been identified as one of the most promising long term UK control strategies ( Krebs (1997) "Bovine Tuberculosis in Cattle & Badgers” HMSO ) and the development of an efficacious vaccine continues to be a research priority.
  • promising vaccines against bovine TB are based on heterologous prime-boost combinations that include the live attenuated M. bovis vaccine strain Bacille Calmette-Guerin (BCG) as one of their components ( Hogarth et al. (2006) J. Pharm. Pharmacol. vol.
  • DIVA reagents have so far been largely realised through their use in blood-based interferon-y (IFN- ⁇ ) release assays (IGRAs).
  • IFN- ⁇ blood-based interferon-y release assays
  • WO2009/060184 and Sidders et al. 2008; Am. J. Microbiol. vol. 76 p 3932-3939 ) disclosed several polypeptides including epitopes from Rv3615c which were found to be useful to detect M. bovis or M. tuberculosis infection, using such an assay. This polypeptide has also been confirmed as useful to detect M. tuberculosis infection in humans ( Millington et al. (2011) Proc. Natl. Acad. Sci. USA vol.
  • Rv3615c is referred to herein using the M. tuberculosis genome annotation (http://genolist.pasteur.fr/TubercuList/, accessed 14 th July 2011). In the M. bovis genome, it is annotated as Mb3645c (http://genolist.pasteur.fr/BoviList/, accessed 14 th July 2011).
  • Whelan et al. (2010; J. Clin. Microbiol. vol. 48 p3176-3181 ) discusses use of ESAT-6, CFP-10, MPB70, MPB83 and Rv3615c in combination as a skin test.
  • WO98/53075 discloses amino acid sequences contained within M. tuberculosis proteins.
  • the present invention accordingly addresses the problem of providing discriminatory diagnostic reagents for the detection of mycobacterial infections, particularly in a DIVA skin-test format.
  • a diagnostic reagent comprising:
  • This reagent elicits a positive result when used in an assay to determine whether an animal has a tuberculosis infection or has been exposed to a tuberculosis agent.
  • the diagnostic reagent can allow a user to differentiate between an animal having a tuberculosis infection and one which has been vaccinated against such an infection, as is disclosed herein for the first time and as will be explained in further detail below.
  • a negative result is also obtained when the animal is unvaccinated and uninfected (or unexposed to a tuberculosis agent).
  • the animal may be a mammal, for example a cow, a badger or a human being.
  • the diagnostic reagent may further comprise amino acid sequence SEQ ID NO:7 or a sequence having at least 85% sequence identity thereto.
  • the diagnostic reagent may alternatively or further comprise at least one polypeptide each comprising at least one of the amino acid sequences SEQ ID NO:1, 2, 3, 4, 5, 6 or 9, or a sequence having at least 85% sequence identity to one of SEQ ID NO:1, 2, 3, 4, 5, 6 or 9.
  • the diagnostic reagent may comprise all of amino acid sequences SEQ ID NOs:1-9. This may be in the form of individual polypeptides each having an amino acid sequence selected from SEQ ID NOs:1-9, or may be one or more fusion proteins each comprising two or more of SEQ ID NOs:1-9. These sequences were included in peptide pool Sec#1 described herein and shown in Table 3 below.
  • tuberculosis infection indicates an infection in which the causative agent is a Mycobacterium , for example, M. tuberculosis , M. bovis , and/or M. africanum. In some cases, the infection can be the result of exposure to a combination of these bacterial species.
  • the term “tuberculosis agent” indicates an organism capable of causing tuberculosic symptoms, typically a Mycobacterium , for example M. tuberculosis, M. bovis , and/or M. africanum.
  • a vaccine which might be administered to an animal to vaccinate against a tuberculosis infection includes the BCG vaccine.
  • the diagnostic reagent may alternatively or further comprise a polypeptide comprising at least one of the amino acid sequences SEQ ID NOs:10-69 or a sequence having at least 85% sequence identity to one of SEQ ID NOs:10-69.
  • SEQ ID Nos: 50-69 were included in peptide pools #11 and #14 described herein and shown in Table 2 below.
  • the diagnostic reagent may comprise SEQ ID NOs:8 and 60-69.
  • the diagnostic reagent may comprise the polypeptides having amino acid sequences SEQ ID NOs:11, 18, 22, 30 and 45, or may comprise the polypeptides having amino acid sequences SEQ ID NOs:13, 15, 24, 46; in other words, the diagnostic reagent may comprise any combination of polypeptides which each have an amino acid sequence freely selected from those indicated by SEQ ID NOs: 11-49.
  • Sequences SEQ ID NOs:11-21 are peptides which are fragments of ESAT-6.
  • Sequences SEQ ID NOs:22-31 are peptides which are fragments of CFP-10.
  • Sequences SEQ ID NOs:32-43 are peptides which are fragments of Rv3615c. These sequences are discussed in co-pending application no. PCT/GB2011/050843 .
  • SEQ ID NOs:44, 45 and 46 are full-length ESAT-6, CFP-10, and Rv3615c, respectively.
  • SEQ ID NO:47 is the protein MPB83
  • SEQ ID NO:48 is a fragment of MPB83
  • SEQ ID NO:49 is the protein MPB70.
  • the diagnostic reagent may comprise SEQ ID NOs:8, 11-42 and 60-69.
  • the diagnostic reagent may comprise individual peptide sequences, or may comprise one or more fusion proteins each comprising at least two amino acid sequences selected from SEQ ID NOs:1-69, preferably comprising at least one of SEQ ID NOs:7 or 8.
  • the diagnostic reagent may be for use in a method of detecting a tuberculosis infection in a mammal, or of detecting exposure of an animal to a tuberculosis agent.
  • the method is capable of confirming such infection or exposure, as differentiated from vaccination.
  • the method may be a skin test such as a caudal fold test (CFT), single intradermal test (SIT) or single intradermal comparative cervical test (SICCT).
  • CFT caudal fold test
  • SIT single intradermal test
  • SICCT single intradermal comparative cervical test
  • the diagnostic reagent may be in the form of a sterile injectable preparation which may be an aqueous or an oleaginous suspension, or a suspension in a non-toxic parenterally-acceptable diluent or solvent.
  • a sterile injectable preparation which may be an aqueous or an oleaginous suspension, or a suspension in a non-toxic parenterally-acceptable diluent or solvent.
  • the aqueous suspension may be prepared in, for example, mannitol, water, Ringer's solution or isotonic sodium chloride solution. Alternatively, it may be prepared in phosphate buffered saline solution.
  • the oleaginous suspension may be prepared in a synthetic monoglyceride, a synthetic diglyceride, a fatty acid or a natural pharmaceutically-acceptable oil.
  • the fatty acid may be an oleic acid or an oleic acid glyceride derivative.
  • the natural pharmaceutically-acceptable oil may be an olive oil, a castor oil, or a polyoxyethylated olive oil or castor oil.
  • the oleaginous suspension may contain a long-chain alcohol diluent or dispersant, for example, Ph. Helv.
  • a diagnostic reagent for use in a method of detecting a tuberculosis infection in a mammal or of detecting exposure of an animal to a tuberculosis agent, and differentiating from vaccination of the animal, the diagnostic reagent comprising amino acid sequence SEQ ID NO:8, wherein the method is a skin test which elicits a positive result when the mammal is infected with a tuberculosis agent and which elicits a negative result when the mammal has been vaccinated against infection with a tuberculosis agent or when the mammal is unvaccinated and/or uninfected.
  • a further aspect of the invention provides a method of detecting a tuberculosis infection in an animal, or exposure of an animal to a tuberculosis agent, comprising the steps of
  • the population of cells may include T-cells.
  • Recognition of the diagnostic reagent by said cells may be by way of, for example, binding of a T cell receptor to the diagnostic reagent, for example, binding of the T cell receptor to at least one polypeptide included in the diagnostic reagent.
  • the method may comprise a cell-mediated immunity (CMI) assay, which may detect interferon gamma (IFN- ⁇ ) as described herein.
  • CMI cell-mediated immunity
  • IFN- ⁇ interferon gamma
  • a further aspect of the invention provides a diagnostic reagent as defined in the first aspect of the invention, for use in a method of detecting a tuberculosis infection in an animal, or exposure of an animal to a tuberculosis agent, comprising the steps of
  • Using" and “use” of polypeptides and diagnostic reagents in a skin test typically involves intradermal injection of the polypeptide(s) and/or diagnostic reagent into the animal.
  • the skin test may be a CFT, SIT or SICCT test, as described in the Office International des Epizooties (OIE) Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (ISBN-10:92-9044-718-4; http://www.oie.int/eng/bod/ mmanual/a_summry.htm, accessed 14th July 2011 ).
  • OIE Office International des Epizooties
  • the methods disclosed herein may be for detecting M. bovis or M. tuberculosis infection in an animal, for example (but not limited to) a mammal such as a cow, badger or human being.
  • a mammal such as a cow, badger or human being.
  • the diagnostic reagents are able to differentiate between an infected animal and a vaccinated animal, the user can be sure that a positive result from the method is a true positive indicating infection, rather than a false positive resulting from earlier vaccination of the animal.
  • the methods in this aspect of the invention provide a method of detecting a tuberculosis infection in an animal, or exposure of an animal to a tuberculosis agent, comprising conducting a skin test on the animal using at least one diagnostic reagent according to the first aspect of the invention, wherein use of the method on an animal which has been vaccinated against infection by a tuberculosis agent results in a negative skin test being obtained and use of the method on an animal infected with a tuberculosis agent results in a positive skin test being obtained.
  • a diagnostic kit comprising a diagnostic reagent according to the first aspect of the invention.
  • the diagnostic kit may be for use in one or more methods disclosed herein.
  • the diagnostic reagent may be in liquid form, as outlined above, or may be in solid (for example, lyophilised) form. It may be included in the kit in the form of at least one aliquot of 0.05-0.15ml containing 1-15 ⁇ g of each polypeptide contained in the diagnostic reagent.
  • the kit may comprise aliquots of about 0.05ml, about 0.06ml, about 0.07ml, about 0.08ml, about 0.09ml, about 0.1ml, about 0.11ml, about 0.12ml, about 0.13ml, about 0.14ml or about 0.15ml, containing 1-15 ⁇ g, 3-12 ⁇ g, 5-10 ⁇ g of each protein, for example, about 5 ⁇ g, about 6 ⁇ g, about 7 ⁇ g, about 8 ⁇ g, about 9 ⁇ g or about 10 ⁇ g of each polypeptide.
  • Each aliquot may be contained in a disposable injection device.
  • the kit may further comprise at least one sample of PPD.
  • the diagnostic reagent is able to detect a tuberculosis infection in an animal, or exposure of an animal to a tuberculosis agent, for example it may be able to detect infection by or exposure to M. bovis or M. tuberculosis.
  • the diagnostic reagent comprises one or more of SEQ ID NOs:1-9 and/or 50-69, it enables a user to differentiate between a tuberculosis infected animal and an animal which has been vaccinated against a tuberculosis infection, by methods such as described herein. As mentioned above, this enables the user to rely on positive test results when using the diagnostic reagent as an indication of tuberculosis infection rather than of vaccination.
  • a polypeptide (which may be isolated) consisting of the amino acid sequence of SEQ ID NO:8, or consisting of between 15-65 amino acids and comprising SEQ ID NO:8.
  • the term "functional variant” indicates a polypeptide in which the amino acid sequence differs from the base sequence from which it is derived in that one or more amino acids within the sequence are substituted for other amino acids.
  • the variant is a functional variant because the functional characteristics of the polypeptide from which the variant is derived are maintained. For example, a similar immune response is elicited by exposure of an animal, or a sample from an animal, to the variant polypeptide as compared to the non-variant.
  • any amino acid substitutions, additions or deletions must not alter or significantly alter the tertiary structure of one or more epitopes contained within the polypeptide from which the variant is derived.
  • the skilled person is readily able to determine appropriate functional variants without the application of inventive skill.
  • Amino acid substitutions may be regarded as "conservative" where an amino acid is replaced with a different amino acid with broadly similar properties. Non-conservative substitutions are where amino acids are replaced with amino acids of a different type.
  • conservative substitution is meant the substitution of an amino acid by another amino acid of the same class, in which the classes are defined as follows: Class Amino acid examples Nonpolar: Ala, Val, Leu, Ile, Pro, Met, Phe, Trp Uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gln Acidic: Asp, Glu Basic: Lys, Arg, His.
  • altering the primary structure of a polypeptide by a conservative substitution may not significantly alter the activity of that polypeptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. This is so even when the substitution is in a region which is critical in determining the peptide's conformation.
  • non-conservative substitutions are possible provided that these do not disrupt the tertiary structure of an epitope within the polypeptide, for example, which do not interrupt the immunogenicity (for example, the antigenicity) of the peptide.
  • the addition or deletion of a small number (for example, up to about 10, or up to about 5, for example about 1, 2, 3, 4 or 5) of amino acids to the Nor C-terminus of a polypeptide may not adversely affect the immunogenicity of the peptide and such variants to an amino acid sequence are particularly envisaged as being included within the term "functional variant" of a polypeptide.
  • variants may be at least about 85%, 90%, 95%, 96%, 97%, 98% or about 99% identical to the base sequence, determined as a percentage of the full length of the longest of the polypeptides being compared.
  • Sequence identity between amino acid sequences can be determined by comparing an alignment of the sequences. When an equivalent position in the compared sequences is occupied by the same amino acid, then the molecules are identical at that position. Scoring an alignment as a percentage of identity is a function of the number of identical amino acids at positions shared by the compared sequences. When comparing sequences, optimal alignments may require gaps to be introduced into one or more of the sequences to take into consideration possible insertions and deletions in the sequences. Sequence comparison methods may employ gap penalties so that, for the same number of identical molecules in sequences being compared, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps.
  • Calculation of maximum percent identity involves the production of an optimal alignment, taking into consideration gap penalties.
  • the percentage sequence identity may be determined using the BLASTP software, publicly available at http://blast.ncbi.nlm.nih.gov/Blast.cgi (accessible on 14th July 2011), using default parameter settings. Comparison should be determined for the full length sequence of the polypeptide, to avoid high sequence identity over a short fragment of the polypeptide.
  • the invention also encompasses fusion proteins comprising more than one of SEQ ID NOs:1-10 and/or 50-69.
  • the full length antigen proteins listed in Table 3 may be excluded (e.g., Rv1038c, Rv1197, Rv1792, etc.).
  • nucleic acid encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO:8, or consisting of between 15-65 amino acids and comprising SEQ ID NO:8.
  • any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.
  • Heparinised blood samples were obtained from naturally infected, SICCT-positive reactors from herds known to have bovine tuberculosis (BTB) as determined by the Animal Health Agency. Heparinised blood samples were also obtained from 4 animals who were experimentally infected ca. 6 months with an M. bovis field strain from Great Britain (AF 2122/97) by intratracheal instillation of 1 X 10 3 CFU as previously described ( Dean et al. (2005) Infect. Immun. vol. 73 pp 6467-6471 ). A detailed post mortem examination of 36 TB-reactor animals revealed visible TB-lesions in all but four animals, confirming the presence of active disease.
  • BTB bovine tuberculosis
  • Heparinised blood samples were obtained from animals vaccinated with BCG as previously described ( Vordermeier et al. (1999) Clin. Diagn. Lab. Immunol. vol. 6 pp 675-682 ). Briefly, calves (ca. 6 months of age) from BTB-free herds were vaccinated with BCG Pasteur by subcutaneous injection of 1 X 10 6 CFU into the side of the neck.
  • peptide pools were dissolved in RPMI 1640 (Gibco, UK) containing 20% dimethyl sulfoxide (DMSO) to obtain a concentration of 1mg/ml/peptide, and the peptide pools were used to stimulate whole blood at a final concentration of 5 ⁇ g/ml/peptide.
  • Peptides that comprised the pools for some antigens were synthesized individually (Mimotopes Pty Ltd, Clayton, Australia), dissolved in RPMI 1640 containing 20% DMSO to obtain a concentration of 5mg/ml and used individually to stimulate whole blood at a final concentration of 10 ⁇ g/ml, or formulated into additional peptide pools at a concentration of 10 ⁇ g/ml/peptide.
  • Peptides from ESAT-6 and CFP-10 were formulated to obtain a peptide cocktail as previously described ( Vordermeier et al. (2001) Clin. Diagn. Lab. Immunol. vol. 8 pp 571-578 ) and were used at a final concentration of 5 ⁇ g/ml/peptide. This peptide cocktail was used as a 'gold standard' with which to compare the immunogenicities of the other antigens.
  • Bovine tuberculin (PPD-B) was supplied by the Tuberculin Production Unit at the Veterinary Laboratories Agency, Weybridge, Surrey, UK and was used at a final concentration of 10 ⁇ g/ml.
  • Staphylococcal enterotoxin B (SEB; Sigma-Aldritch, UK) was included as a positive control at a final concentration of 1 ⁇ g/ml, while whole blood was incubated with RPMI 1640 alone as a negative control.
  • Antigens were administered at 8 sites per animal (4 on each side of the neck). Antigens were administered in a volume of 100 ⁇ l. All defined protein or peptide antigen cocktails were administered at a concentration of 5ug/ml per cocktail component. The skin thickness was measured at the injection site immediately prior to intradermal administration of antigen. Skin thickness at the injection site was re-measured 72 hours after antigen administration and the increase in skin-thickness over this time is determined. This method is also suitable for determining the reaction in cattle uninfected with M. bovis, whether vaccinated or unvaccinated against such infection.
  • Purified recombinant protein antigens were supplied by Lionex GmbH.
  • the composition of the cocktail of ESAT-6, CFP-10 and Rv3615c was SEQ ID NOs:11-43.
  • a combination of 11 peptides providing a complete sequence overlap for each of the antigens Rv3020c and Rv2346c was used, as listed in Table 2 below (SEQ ID NOs:7 and 50-59 provide complete sequence overlap for Rv2346c and SEQ ID NOs:8 and 60-69 provide complete sequence overlap for Rv3020c).
  • the Rv3020c and Rv2346c cocktails i.e.
  • peptide pools #14 and #11, respectively, as shown in Table 2) were tested individually and in combination with a cocktail containing the overlapping peptides of ESAT-6, CFP-10 and Rv3615c (i.e., SEQ ID NOs:11-43), which is itself the subject of co-pending patent application PCT/GB2011/050843 .
  • M. bovis secreted proteins are likely to contain immunogenic antigens that can be used to increase the specificity of diagnostic tests.
  • all TB-reactor and BCG-vaccinated animals responded to PPD-B and to the positive control antigen SEB, whilst 22 TB-reactor animals (96%) and 2 BCG-vaccinated animals (25%) responded to the ESAT-6/CFP-10 peptide cocktail (data not shown).
  • Figure 1 details the responder frequencies for the top 8 most frequently recognised peptide pools, i.e. those that induced an IFN- ⁇ response in more than half of the TB-reactor animals studied. Strikingly, all but one peptide pool (#30-2) represented antigens belonging to the ESAT-6 protein family. Interestingly, peptide pools #11 and #14 were not recognized by any of the BCG-vaccinated animals, suggesting that they contain peptides with potential application as DIVA reagents. The peptides included in these pools are indicated in Table 2. Table 2: Sequences of peptide pools #11 and #14.
  • a peptide pool (Sec#1) consisting of 10 peptides was constructed. Firstly, peptides #42 and #55 (SEQ ID NOs:7 & 8, respectively) were selected as they were the two most frequently recognised peptides (responder frequencies of 50% and 36% respectively) and also because they belonged to peptide pools not recognised by BCG-vaccinated animals (pools #11 and #14 respectively, Figure 1 ).
  • a further four peptides (peptides #20, #29, #33 and #64) were next selected as they were recognised in TB-reactor animals that failed to respond to peptides #42 or #55 (data not shown).
  • a further four peptides (peptides #16, #19, #25 and #57) were included due to their location in regions of homology between multiple ESAT-6 proteins (see Table 3).
  • the responder frequency to the Sec#1 peptide pool was significantly greater in TB-reactor animals (p ⁇ 0.05, Fisher's Exact Test), with 14 out of 22 (64%) TB-reactor animals recognising the peptide pool compared with 6 out of 21 (29%) BCG-vaccinated animals.
  • the individual peptide components of the Sec#1 peptide pool were rescreened for their ability to induce an IFN- ⁇ response in BCG-vaccinated animals.
  • the skin-test data is shown in Figure 4 .
  • Addition of the peptide cocktail of Rv3020c (SEQ ID NOs:8 and 60-69) to the reference cocktail containing peptides of ESAT-6, CFP-10 and Rv3615c (SEQ ID NOs: 11-43) demonstrated significantly stronger responses than the reference cocktail alone, see Figure 5 .
  • the Sec#2 cocktail contained several immunodominant peptides with restricted expression amongst the ESAT-6 proteins; for example, peptide #55 is located only within Rv3020c while peptide #42 is located in Rv2346c and Rv1793 (Table 3). However, given the high degree of amino acid similarity between the members of the ESAT-6 protein family, several of these peptide sequences represented multiple antigens. For example, peptides #16 and #20 are located in Rv1038c, Rv1197, Rv1792, Rv2347c and Rv3620c, while peptide #33 is located in Rv1198, Rv2346c, Rv3619c, Rv1037c and Rv1793.
  • targeting these shared sequences not only reduces the number of different components within the DIVA reagent but may also exploit a potential greater antigenic load for these regions.
  • the ESAT-6/CFP-10 peptide cocktail used in the studies presented herein has been developed as a DIVA reagent in cattle, with reported sensitivities of approximately 78% in M. bovis -infected animals ( Sidders et al. (2008) Infect. Immun. vo. 76 pp 3932-3939 ; Vordermeier et al. (2001) Clin. Diagn. Lab. Immunol. vol. 8 pp 571-578 ).
  • one area of research of high importance is the identification of reagents that may complement the ESAT-6/CFP-10 peptide cocktail in the diagnosis of bovine TB. Recently, the inventors have demonstrated that 4 out of 7 (57%) M.

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Claims (15)

  1. Réactif de diagnostic comprenant :
    (i) un polypeptide constitué par une séquence d'acides aminés SEQ ID NO : 8 ; ou
    (ii) un polypeptide constitué par 15 à 65 acides aminés de la SEQ ID NO : 8 ;
    le réactif caractérisé en ce qu'il suscite un résultat de dosage de diagnostic négatif quand un dosage de l'infection par la tuberculose est réalisé sur un échantillon d'un animal qui a été vacciné contre l'infection par un agent de la tuberculose.
  2. Réactif de diagnostic selon la revendication 1 comprenant en outre :
    (i) la séquence d'acides aminés SEQ ID NO : 7 ou une séquence ayant au moins 85 % d'homologie de séquence avec celle-ci ; et/ou
    (ii) comprenant en outre au moins une des séquences d'acides aminés SEQ ID NO : 1, 2, 3, 4, 5, 6 ou 9 ou une séquence ayant au moins 85 % d'homologie de séquence avec l'une des SEQ ID NO : 1, 2, 3, 4, 5, 6 ou 9 ; et/ou
    (iii) comprenant toutes les SEQ ID NO : 1 à 9.
  3. Réactif de diagnostic selon la revendication 1 ou 2 comprenant en outre au moins une des séquences d'acides aminés SEQ ID NO : 10 à 69 ou une séquence ayant au moins 85 % d'homologie de séquence avec l'une des SEQ ID NO : 10 à 69.
  4. Réactif de diagnostic selon la revendication 1 comprenant en outre les séquences d'acides aminés SEQ ID NO : 60 à 69, comprenant facultativement les séquences d'acides aminés SEQ ID NO : 7, 11 à 42 et 50 à 69.
  5. Réactif de diagnostic destiné à être utilisé dans un procédé de détection d'une infection par la tuberculose chez un mammifère ou de détection de l'exposition d'un animal à un agent de la tuberculose, et de différenciation de la vaccination de l'animal, le réactif de diagnostic comprenant la séquence d'acides aminés SEQ ID NO : 8, dans lequel le procédé est un test cutané qui suscite un résultat positif quand le mammifère est infecté avec un agent de la tuberculose et qui suscite un résultat négatif quand le mammifère a été vacciné contre l'infection avec un agent de la tuberculose ou quand le mammifère est non vacciné et/ou non infecté.
  6. Procédé de détection d'une infection par la tuberculose chez un animal, ou de l'exposition d'un animal à un agent de la tuberculose, comprenant les étapes
    (i) de mise en contact in vitro d'une population de cellules de l'animal avec au moins un réactif de diagnostic selon l'une quelconque des revendications 1 à 4 ; et
    (ii) de détermination si les cellules de ladite population reconnaissent le réactif de diagnostic ; facultativement dans lequel la population de cellules inclut des cellules T.
  7. Procédé selon la revendication 6, comprenant un dosage d'immunité à médiation cellulaire (IMC), facultativement dans lequel le dosage IMC détecte l'interféron gamma (IFN-γ).
  8. Réactif de diagnostic selon l'une quelconque des revendications 1 à 4 destiné à être utilisé dans un procédé de détection d'une infection par la tuberculose chez un animal, ou de l'exposition d'un animal à un agent de la tuberculose, comprenant les étapes
    (i) de mise en contact in vivo d'une population de cellules de l'animal avec le réactif de diagnostic ; et
    (ii) de détermination si les cellules de ladite population reconnaissent le réactif de diagnostic.
  9. Trousse de diagnostic comprenant un réactif de diagnostic selon l'une quelconque des revendications 1 à 4.
  10. Trousse selon la revendication 9 dans laquelle le réactif de diagnostic est capable de détecter une infection par la tuberculose chez un animal, ou l'exposition d'un animal à un agent de la tuberculose.
  11. Trousse selon la revendication 10 dans laquelle le réactif de diagnostic est selon la revendication 1 ou 2 et est adapté à une utilisation pour faire la différence entre un animal ayant une infection par la tuberculose et un animal vacciné contre l'infection par la tuberculose.
  12. Polypeptide constitué par une séquence d'acides aminés de la SEQ ID NO : 8, ou constitué par 15 à 65 acides aminés et comprenant la SEQ ID NO : 8.
  13. Acide nucléique codant pour un polypeptide constitué par au moins un polypeptide selon la revendication 12.
  14. Vecteur comprenant au moins un acide nucléique selon la revendication 13.
  15. Cellule transformée avec le vecteur selon la revendication 14.
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WO2001004151A2 (fr) * 1999-07-13 2001-01-18 Statens Serum Institut Vaccins et produits de diagnostic pour la tuberculose derives de la famille genique mycobacterium tuberculosis esat-6
WO2001062893A2 (fr) * 2000-02-25 2001-08-30 Corixa Corporation Composes et methodes de diagnostic et d'immunotherapie de la tuberculose
US6613881B1 (en) * 1997-05-20 2003-09-02 Corixa Corporation Compounds for immunotherapy and diagnosis of tuberculosis and methods of their use

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US6555653B2 (en) * 1997-05-20 2003-04-29 Corixa Corporation Compounds for diagnosis of tuberculosis and methods for their use
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US6613881B1 (en) * 1997-05-20 2003-09-02 Corixa Corporation Compounds for immunotherapy and diagnosis of tuberculosis and methods of their use
WO2001004151A2 (fr) * 1999-07-13 2001-01-18 Statens Serum Institut Vaccins et produits de diagnostic pour la tuberculose derives de la famille genique mycobacterium tuberculosis esat-6
WO2001062893A2 (fr) * 2000-02-25 2001-08-30 Corixa Corporation Composes et methodes de diagnostic et d'immunotherapie de la tuberculose

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US20130203087A1 (en) 2013-08-08
US20170097349A1 (en) 2017-04-06
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ES2600907T3 (es) 2017-02-13
US20150093766A1 (en) 2015-04-02
EP2596357A2 (fr) 2013-05-29
ES2724730T3 (es) 2019-09-13
US10309962B2 (en) 2019-06-04

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